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. 2022 Apr 6;53(3):1231–1240. doi: 10.1007/s42770-022-00740-2

The antifungal and antibiofilm activity of Cymbopogon nardus essential oil and citronellal on clinical strains of Candida albicans

Leonardo Antunes Trindade 1,✉, Laísa Vilar Cordeiro 2, Daniele de Figuerêdo Silva 2, Pedro Thiago Ramalho Figueiredo 2, Marcela Lins Cavalcanti de Pontes 2, Edeltrudes de Oliveira Lima 2, Alessandra de Albuquerque Tavares Carvalho 1
PMCID: PMC9433487  PMID: 35386096

Abstract

Objective

This study investigated the antifungal and antibiofilm activity of Cymbopogon nardus essential oil (EO) and its major compound, citronellal, in association with miconazole and chlorhexidine on clinical strains of Candida albicans. The likely mechanism(s) of action of C. nardus EO and citronellal was further determined.

Materials and methods

The EO was chemically characterized by gas chromatography coupled with mass spectrometry (GC-MS). The antifungal activity (MIC/MFC) and antibiofilm effects of C. nardus EO and citronellal were determined by the microdilution method, and their likely mechanism(s) of action was determined by the sorbitol and ergosterol assays. Then, the samples were tested for a potential association with standard drugs through the checkerboard technique. Miconazole and chlorhexidine were used as positive controls and the assays were performed in triplicate.

Results

The GC-MS analysis tentatively identified citronellal as the major compound in C. nardus EO. Both samples showed antifungal activity, with MIC of 256 µg/mL, as compared to 128 µg/mL and 8 µg/mL of miconazole and chlorhexidine, respectively. C. nardus EO and citronellal effectively inhibited biofilm formation (p < 0.05) and disrupted preformed biofilms (p < 0.0001). They most likely interact with the cell membrane, but not the cell wall, and did not present any synergistic activity when associated with standard drugs.

Conclusion

C. nardus EO and citronellal showed strong in vitro antifungal and antibiofilm activity on C. albicans.

Clinical relevance

Natural products have been historically bioprospected for novel solutions to control fungal biofilms. Our data provide relevant insights into the potential of C. nardus EO and citronellal for further clinical testing. However, additional bioavailability and toxicity studies must be carried out before these products can be used for the chemical control of oral biofilms.

Keywords: Cymbopogon, Citronellal, Candida, Antifungal, Antibiofilm

Introduction

Candida albicans is an opportunistic microorganism in the oral microbiome that may transition into a pathogenic state and cause infections in immunologically susceptible individuals [1]. One of the main virulence attributes of yeasts is their ability to form biofilms, which is associated with phenotypic changes, morphological diversification, and drug resistance [2]. The interaction between C. albicans and host cells is characterized by a complex expression of virulence factors for yeast adhesion, invasion, and tissue damage [3], including morphological plasticity, thymotropism (contact detection), expression of adhesins and invasins on the cell surface, secretion of hydrolytic enzymes, and biofilm development [4].

Fungal biofilms are highly dynamic heterogeneous communities formed by hyphae, pseudohyphae, and blastoconidia, embedded in an extracellular matrix containing polysaccharides, proteins, and nucleic acids [5]. Environmental factors (e.g., saliva, gingival fluid, pH, and nutrients) favor the coaggregation and coadhesion between yeasts and other microorganisms, especially bacteria, which contributes to the onset of biofilm-dependent diseases such as dental caries and periodontal disease [6].

In healthy individuals, the presence of other commensals and the immune response restrict the overgrowth of C. albicans [7]. However, under immunosuppressed conditions, C. albicans burden may lead to symptoms from mild local discomfort and changes in taste sensation to severe systemic infections with significant morbidity and mortality [8].

Fungal biofilms are more resistant to host’s defense mechanisms and conventional antimicrobial therapy, resulting in severe and persistent infections, undesirable side effects, and rapid development of drug resistance [9–12]. Previous studies demonstrated that approximately 40% of C. albicans strains were resistant to miconazole; 45% of them were resistant to fluconazole in individuals who had received prior therapy, and among the resistant isolates, 93% had developed cross-resistance to itraconazole [13, 14].

Broad-spectrum antibiotic therapy and poor denture disinfection, associated with hyposalivation, can favor the onset of oral candidiasis [15, 16]. Polyenes (amphotericin B and nystatin) and azoles (fluconazole, itraconazole, miconazole, ketoconazole) are the drugs available for the treatment of fungal infections. Azoles are generally fungistatic and considered to be the first choice of treatment [17]. Chlorhexidine is also a broad-spectrum antimicrobial that inhibits biofilm formation of both bacteria and yeasts [18].

Natural products have been historically bioprospected as an alternative to the use of synthetic chemicals. The genus Cymbopogon consists of eighty-five species and many of which are known to have medicinal properties. Cymbopogon nardus L. Rendle is a plant originated from Ceylon and India and belongs to the Poaceae family, subfamily Panicoideae [19]. The essential oil (EO) of C. nardus, popularly known as citronella, was reported to be an insect repellent [20], antispasmodic, rubefacient, stimulant, carminative, diaphoretic [21], and antimicrobial [22–26]. It is mainly composed of monoterpenes (±80%) such as citronellal, citronellol, and geraniol [20].

Thus, in our study, we investigated the antifungal and antibiofilm activity of C. nardus EO and its major compound, citronellal, in association with miconazole and chlorhexidine on clinical strains of Candida albicans.

Materials and methods

C. nardus EO and citronellal samples

C. nardus (L.) Rendle EO and the phytoconstituent citronellal were purchased from BIOESSÊNCIA® and SIGMA-ALDRICHT®. Miconazole and chlorhexidine were obtained from SIGMA-ALDRICHT® and RIOQUÍMICA®—Industria Farmacêutica, respectively [17, 18]. The culture medium RPMI 1640 was obtained from HIMEDIA® and prepared following the manufacturer’s instructions.

Microorganisms and growth conditions

Biological tests were performed at the Laboratory of Clinical Mycology of the Department of Pharmaceutical Sciences, Federal University of Paraíba (Brazil). C. albicans ATCC 76485 and clinical isolates recovered from the oral cavity and dentures were used, namely, LM 2B, LM 4, LM 9B, LM 9P, LM 12B, LM 12P, LM 13B, LM 19B, LM 19P, LM 62, LM 108, and LM 122. During the assays, the strains were kept in Sabouraud dextrose agar (DIFCO®), stored at 4 °C and room temperature (28 to 30 °C). A clinical strain (LM 4) was randomly selected for comparison with the standard strain (ATCC 76485).

For the inoculum, the selected strains were kept in the culture media for 24–48 h at 35–37 °C and adjusted in a sterile saline solution to a barium sulfate suspension in the tube number 0.5 of the McFarland scale. After vortexing (Fanem) for 2 min, the suspensions were adjusted to 90% transmittance in a spectrophotometer (Leitz-photometer 340-800) to contain ~ 1.0 × 106 CFU/mL [27–29].

Gas chromatography coupled with mass spectrometry (GC/MS) analysis

The EO was chemically characterized by gas chromatography coupled with mass spectrometry (GC/MS). The GC/MS 5975C Series (Agilent Technologies, Palo Alto, USA) was used in a quadrupole system equipped with a nonpolar DB-5 column (Agilent J&W; 60 m long, 0.25 mm internal diameter, 0.25 µm film thickness). The GC oven temperature was kept at 60 °C for 3 min, then increased to 240 °C (2.5 °C min−1) and maintained for 10 min. The pressure of the helium gas flow remained constant at 100 kPa; the injector was set at 250 °C in split mode (1:50). The detector operated at 280 °C, and the ionization potential was set at 70 eV with a scanning speed of 0.5 scans−1 in the m/z 20–350 range.

Minimum inhibitory concentration (MIC)

The MIC of C. nardus EO, citronellal, miconazole, and chlorhexidine was determined through the microdilution technique. Initially, 100 μL of RPMI 1640 was dispensed into the wells of a microdilution plate. Then, 100 μL of the test substance was added at an initial concentration of 1,024 μg/mL and serially diluted at a ratio of 2. Sample concentrations ranged from 1,024 to 2 μg/mL. Lastly, 10 μL aliquots of the inoculum were dispensed into the wells of each column. In parallel, strain viability and susceptibility to miconazole and chlorhexidine were also controlled. The tests were performed in triplicate and the plates were incubated at 35–37 °C for 24–48 h. The MIC was considered as the lowest concentration of the sample that inhibited visible microbial growth [27–29].

Minimum fungicidal concentration (MFC)

Aliquots from the wells corresponding to the MIC and higher concentrations (2×MIC and 4×MIC) were subcultured in 96-well microdilution plates with RPMI 1640 medium to determine the minimum fungicidal concentration (MFC). After 24–48 h of incubation at 35–37 °C, visual readings were performed based on the growth of controls. The MFC was considered as the lowest concentration of the sample that inhibited the growth of the subculture [29].

The action/interaction of C. nardus EO and citronellal on the yeast cell wall and membrane

To investigate the action of C. nardus EO and citronellal on the yeast cell wall, their MICs were determined in the absence and presence of 0.8 M sorbitol in 96-well plates. Briefly, 100 µL of RPMI 1640 supplemented with sorbitol—2-fold concentrated (molecular weight of 182.17 g) (VETEC Química Fina Ltda—Rio de Janeiro/RJ), was added in each well. Then, 100 µL of the test substances (2-fold concentrated) was added to the wells of the first row and serially diluted at a ratio of 2. Then, 10 µL of C. albicans inoculum (ATCC 76485 and LM 4) was added. Growth and sterility controls were performed. The readings were taken after 24–48 h of incubation at 35–37 °C. The tests were performed in triplicate [30].

To assess the interaction of C. nardus EO and citronellal with the yeast cell membrane, their MICs were determined in the absence and presence of ergosterol (Sigma-Aldrich®) at 400 µg/mL. Growth and sterility controls were performed, and the plates were read after 24–48 h of incubation at 35–37 °C [31].

Association with standard drugs by the checkerboard technique

To determine the association of C. nardus EO and citronellal with miconazole and chlorhexidine, the fractional inhibitory concentration (FIC) index was calculated. Briefly, 100 μL of RPMI 1640 was distributed in the wells of a sterile 96-well microplate. Subsequently, 50 μL of the EO and citronellal and 50 μL of the standard drugs were added vertically and horizontally, respectively, at different concentrations (4×MIC, 2×MIC, MIC, MIC/2, MIC/4). Lastly, 20 μL of the adjusted yeast suspension (ATCC 76485 and LM 4) was added and the microplates were incubated at 35–37 °C for 24–48 h [32]. In parallel, media sterility and strain viability were controlled. The tests were carried out in triplicate.

The FIC was calculated through the formula FICA + FICB, where A represents the standard drug and B is the test substance. FICA was calculated by the combined MICA/isolated MICA ratio, and FICB = combined MICB/isolated MICB. The index was interpreted according to the following criteria: synergism (≤0.5), additivity/indifference (>0.5 to ≤4), or antagonism (>4.0) [33].

Inhibition of biofilm formation

The inhibitory effects of C. nardus EO and citronellal on biofilm formation were evaluated in triplicate as described by Balasubramanian et al. [34], with modifications. Briefly, 100 µL of RPMI 1640 medium containing different concentrations (4×MIC, 2×MIC, MIC, MIC/2, MIC/4) of the test substances (EO, citronellal, miconazole, and chlorhexidine) and 10 µL of C. albicans inoculum (ATCC 76485 and LM 4) were added to a 96-well microplate. For the negative control, only the culture medium and inoculum were used. After 48 h of static incubation at 35–37 °C, the contents of the wells were properly discarded and gently washed with sterile distilled water to remove non-adhered cells. The wells were dried at room temperature and 140 µL of 1% crystal violet (NEWPROV®) was transferred. After 40 min, the dye was discarded and its excess was removed from the walls with distilled water. Once the wells were dry, 140 µL of absolute ethanol (RIOQUÍMICA®) was added for 30 min. The plate was read in a microplate spectrophotometer (Multiskan GO) at 590 nm.

Inhibition of preformed biofilms

The capacity of the test substances to disrupt preformed C. albicans biofilms was evaluated in triplicate following the methodology described by Rajasekharan et al. [35], with modifications. In microdilution plates, 10 µL of yeast inoculum (ATCC 76485 and LM 4) and 100 µL of RPMI 1640 medium were statically incubated for 48 h at 35–37 °C for biofilm formation. Then, the content of the wells was discarded and 100 µL of RPMI 1640 medium was added again containing different concentrations of the test substances (4×MIC, 2×MIC, MIC, MIC/2, MIC/4). The plates were incubated statically at 35–37 °C for 48 h. For the negative control, only the culture medium and inoculum were used. Biofilms were stained with 1% crystal violet and solubilized in absolute ethanol, and the plates were read in a microplate spectrophotometer (Multiskan GO) at 590 nm. The percent inhibition of preformed biofilms was calculated using the following formula: % biofilm disruption = [(control ABS590 − test ABS590)/control ABS590] × 100.

Statistical analysis

Data were analyzed by two-way analysis of variance (ANOVA) with Tukey’s post-test, considering a 95% confidence interval and 5% significance level. The statistical analysis was carried out in the GraphPad Prism 8 software.

Results

Chromatographic profile and GC-MS identification of compounds

As shown in Table 1, the GC-MS analysis indicated that citronellal was the major compound in C. nardus EO (42.28%), followed by geraniol (16.78%) and citronellol (11.5%).

Table 1.

Characterization of Cymbopogon nardus essential oil by gas chromatography coupled with mass spectrometry (GC/MS)

Peak# R. time Area Area% Name
1 11.325 325607 0.09 Not identified
2 12.642 15117106 4.11 Limonene
3 12.743 287633 0.08 Not identified
4 13.004 41406524 11.25 2-Pyrrolidinone, 1-methyl-
5 13.646 1730599 0.47 Not identified
6 14.893 212980 0.06 Terpinolene
7 15.364 1731847 0.47 Linalool
8 16.480 96402 0.03 Not identified
9 16.982 5347603 1.45 Neoisopulegol
10 17.311 155568866 42.28 Citronellal
11 17.513 450454 0.12 Not identified
12 17.837 223762 0.06 Not identified
13 18.685 122632 0.03 Not identified
14 19.224 319936 0.09 n-Decanal
15 19.988 42297898 11.50 Citronellol
16 20.446 1090004 0.30 Neral
17 20.899 61746820 16.78 Geraniol
18 21.475 2805738 0.76 Geranial
19 24.210 4844686 1.32 Citronellyl acetate
20 24.622 388495 0.11 Eugenol
21 25.213 4013313 1.09 Geranyl propanoate
22 25.466 4508161 1.23 Elemene<beta>
23 26.331 236988 0.06 Not identified
24 27.398 209456 0.06 Not identified
25 28.111 363957 0.10 Murolene<gamma>
26 28.250 3380216 0.92 Germacrene D
27 28.675 347941 0.09 Amorpha-4,7(11)-diene
28 28.834 1032592 0.28 Muurolene<alpha>
29 28.983 903603 0.25 Germacrene A
30 29.247 1199753 0.33 g-Cadinene
31 29.524 4143162 1.13 d-Cadinene
32 29.947 152199 0.04 a-Maalinene
33 30.286 4817409 1.31 Elemol
34 31.057 3169913 0.86 Germacrene D-4-ol
35 32.675 326613 0.09 Not identified
36 32.950 828952 0.23 Not identified
37 33.066 196126 0.05 Not identified
38 33.193 328783 0.09 Not identified
39 33.309 1254242 0.34 Not identified
40 33.691 203479 0.06 Viridiflorene
41 38.974 192027 0.05 Not identified
367924477 100.02
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Minimum inhibitory and fungicidal concentrations (MIC/MFC)

The MIC and MFC values of the test substances against C. albicans strains ranged between 512 and 2 µg/mL (Table 2). Both C. nardus EO and citronellal showed antifungal activity, with MIC of 256 µg/mL, as compared to 128 µg/mL and 8 µg/mL of miconazole and chlorhexidine, respectively.

Table 2.

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of Cymbopogon nardus essential oil, citronellal, miconazole, and chlorhexidine on Candida albicans strains

Candida albicans strains C. nardus Citronellal Miconazole Chlorhexidine
MIC MFC MIC MFC MIC MFC MIC MFC
ATCC 76485 256 512 128 256 2 2 2 2
LM 2B 64 64 64 128 2 2 4 4
LM 4 64 64 128 256 16 16 4 4
LM 9B 128 64 64 128 2 8 4 4
LM 9P 128 128 128 128 2 4 2 2
LM 12B 128 128 128 256 2 2 4 4
LM 12P 64 64 32 32 2 2 2 2
LM 13B 64 64 128 512 64 256 4 4
LM 19B 64 64 64 128 32 32 4 4
LM 19P 128 256 64 128 32 32 4 4
LM 62 128 256 64 256 128 512 8 8
LM 108 128 128 256 256 16 16 4 4
LM 122 128 512 256 256 4 4 2 2
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Values expressed in µg/mL

The action of C. nardus essential oil and citronellal on the yeast cell wall and membrane

To investigate the effects of C. nardus EO and citronellal on the cell wall and membrane of C. albicans ATCC 76485 and LM 4, their MIC was determined in the absence and presence of sorbitol (0.8 M) and exogenous ergosterol (400 µg/mL). The MIC values of C. nardus EO and citronellal were unaltered in the presence of the osmotic protector (sorbitol). However, they increased 64-fold in the presence of ergosterol, suggesting that their likely mechanism of action is related to the cell membrane (Table 3).

Table 3.

Effect of C. nardus essential oil and citronellal on C. albicans strains (ATCC 76485 and LM 4) in the absence and presence of sorbitol 0.8 M and ergosterol 400 µg/mL

Strains C. nardus Citronellal
MIC (µg/mL) MIC (µg/mL)
Absence of sorbitol Presence of sorbitol Absence of sorbitol Presence of sorbitol
C. albicans ATCC 76485 256 256 128 128
C. albicans LM 4 64 64 128 128
Absence of ergosterol Presence of ergosterol Absence of ergosterol Presence of ergosterol
C. albicans ATCC 76485 256 16384 128 8192
C. albicans LM 4 64 4096 128 8192
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MIC, minimum inhibitory concentration

Checkerboard data

The FIC data showed that more than half of the test associations (n = 5) of C. nardus EO and citronellal with miconazole and chlorhexidine were interpreted as additivity/indifference. This indicates a lack of interaction between them. The other associations (n = 3) showed antagonism, indicating that the effects of one or both substances were smaller in association than individually (Table 4).

Table 4.

Association of Cymbopogon nardus essential oil and citronellal with miconazole and chlorhexidine to determine the fractional inhibitory concentration index (FICI)—checkerboard method

Candida albicans strains C. nardus—miconazole C. nardus—chlorhexidine Citronellal—miconazole Citronellal—chlorhexidine
FIC
ATCC 76485 2.06 1.06 8.06 1.06
LM 4 0.56 4.06 8.06 2.06
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Synergism (≤0.5), additivity/indifference (>0.5 to ≤4), or antagonism (>4.0)

Inhibitory effects on biofilm formation and preformed biofilms

Overall, our findings indicated that C. nardus EO, citronellal, and the positive controls (miconazole and chlorhexidine) effectively inhibited biofilm formation and disrupted preformed biofilms of C. albicans ATCC 76485 and LM 4.

C. nardus EO, citronellal, and chlorhexidine inhibited the development of C. albicans ATCC 76485 biofilms, with no difference between them (p > 0.05) and with significant differences compared to miconazole (p < 0.05) (Graph 1). Likewise, C. nardus EO and citronellal inhibited LM 4 biofilm formation at all tested concentrations, with no significant difference between them nor compared to miconazole and chlorhexidine (p > 0.05). However, significant differences in biofilm inhibition were observed between chlorhexidine and miconazole groups (Graph 2).

Graph. 1.

Graph. 1

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Inhibition of biofilm formation against Candida albicans ATCC 76485 at different concentrations. **p < 0.01 (chlorhexidine vs miconazole); *p < 0.05 (C. nardus vs miconazole and citronellal vs miconazole)

Graph 2.

Graph 2

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Inhibition of biofilm formation against Candida albicans LM 4 at different concentrations. **p < 0.01 (chlorhexidine vs miconazole)

Our data further demonstrated that treatment with C. nardus EO, citronellal, and chlorhexidine disrupted preformed biofilms of C. albicans ATCC 76485 and was more effective than that with miconazole at MIC/4, MIC/2, and MIC (p < 0.0001). At these concentrations, chlorhexidine was more effective than citronellal and the EO (p < 0.05). However, at 2×MIC and 4×MIC, there was no statistically significant difference between them (p > 0.05). C. nardus EO and citronellal disrupted C. albicans ATCC 76485 preformed biofilms at all tested concentrations, with no significant difference between them (p > 0.05) (Graph 3).

Graph. 3.

Graph. 3

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Biofilm formation against Candida albicans ATCC 76485 at different concentrations. MIC/4—s****p < 0.0001 (chlorhexidine vs miconazole; citronellal vs miconazole; C. nardus vs miconazole); ***p < 0.001 (chlorhexidine vs citronellal); **p < 0.01 (chlorhexidine vs C. nardus); MIC/2—****p < 0.0001 (chlorhexidine vs miconazole; citronellal vs miconazole; C. nardus vs miconazole); **p < 0.01 (chlorhexidine vs citronellal; chlorhexidine vs C. nardus); MIC—****p < 0.0001 (chlorhexidine vs miconazole; citronellal vs miconazole; C. nardus vs miconazole); *p < 0.05 (chlorhexidine vs citronellal; chlorhexidine vs C. nardus)

Similarly, treatment with C. nardus EO, citronellal, and chlorhexidine was more effective against LM 4 preformed biofilms than that with miconazole at MIC/4 and MIC/2 (p < 0.0001). Chlorhexidine was more effective than citronellal and the EO at MIC/4 and MIC/2 (p < 0.05) and miconazole at MIC, 2×MIC, and 4×MIC (p < 0.05). No significant differences in the inhibition of preformed biofilms were observed between C. nardus EO, citronellal, and miconazole (MIC, 2×MIC, and 4×MIC) and chlorhexidine (2×MIC and 4×MIC). C. nardus EO and citronellal disrupted C. albicans LM 4 preformed biofilms at all tested concentrations, with no significant difference between them (p > 0.05) (Graph 4).

Graph. 4.

Graph. 4

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Biofilm formation against Candida albicans LM 4 at different concentrations. MIC/4—****p < 0.0001 (chlorhexidine vs miconazole; citronellal vs miconazole; C. nardus vs miconazole; chlorhexidine vs citronellal; chlorhexidine vs C. nardus); MIC/2—****p < 0.0001 (chlorhexidine vs miconazole; citronellal vs miconazole; C. nardus vs miconazole; chlorhexidine vs citronellal); ***p < 0.001 (chlorhexidine vs C. nardus); MIC—****p < 0.0001 (chlorhexidine vs miconazole); ***p < 0.001 (chlorhexidine vs citronellal); **p < 0.01 (chlorhexidine vs C. nardus); MIC×2—*p < 0.05 (chlorhexidine vs miconazole); MIC×4—*p < 0.05 (chlorhexidine vs miconazole)

Discussion

The chemical composition of C. nardus EO can vary according to the geographical origin, stage of plant development, weather conditions, and the part of the plant used to obtain the EO [36]. Several studies have shown that the terpenoid citronellal is the major phytoconstituent in C. nardus EO [37–40]. This is consistent with our GC-MS data indicating that citronellal accounted for 42.28% of the EO composition.

Our study demonstrated that C. nardus EO and citronellal were able to inhibit 100% C. albicans growth at 512 µg/mL. Other studies reported that these concentrations can range from 128 to 1,024 µg/mL [26, 41], which could be due to differences in the EO chemical composition and strain susceptibility. Chlorhexidine presented the best MIC and MFC results, but when used chronically, it can cause adverse effects such as taste alterations, oral peeling, unpleasant taste, and tooth pigmentation [42, 43]. Thus, the use of alternative drugs or formulations with similar or greater effectiveness, better tolerability, and less adverse effects is much desired.

Our findings suggest that C. nardus EO and citronellal most probably act in the yeast cell membrane due to the considerable increase in their MIC values in the presence of ergosterol. Ergosterol is one of the main components of C. albicans cell membrane that may complex with other molecules and increase permeability. Previous studies showed a remarkable decrease in ergosterol levels in the presence of citronellal, which further supports our data [44, 45]. Generally, lipophilic compounds present in EOs act by breaking or disrupting the cell membrane structure, causing the loss of several enzymes and nutrients [46, 47].

The checkerboard technique has been extensively used to find significant associations in vitro between antimicrobials with distinct mechanisms of action [48]. This approach aims to expand the antimicrobial spectrum, prevent the emergence of resistant strains, and minimize toxicity [49]. The association of standard drugs with natural products can enhance or inhibit the effects of the former, or have no effects whatsoever [50]. In our study, the association of the EO and citronellal with miconazole and chlorhexidine yielded indifferent or antagonistic interactions, demonstrating that it is not beneficial for therapeutic use, probably because they have similar mechanisms of action.

C. albicans and non-albicans biofilms are often resistant to commercial antifungal drugs [51–53], and drug-associated biofilm resistance is considered a multifactorial phenomenon. A susceptibility study showed that of 652 isolated clinical strains of C. albicans, 41.10% were resistant to miconazole and 35.74% to nystatin [13]. Our results showed that C. nardus EO and citronellal showed remarkable antibiofilm activity against developing and preformed biofilms. These findings are in line with the study by Guiotti et al. [54], who demonstrated that C. nardus-based disinfection solutions were effective in killing C. albicans biofilms.

The main resistance mechanisms responsible for decreasing the susceptibility of Candida biofilms to antifungals are drug sequestration by the extracellular polymeric matrix (EPM), increased drug efflux, high cell density, presence of persistent cells, and the activation of the stress-responsive pathway [55–57]. Some EOs exhibit excellent antimicrobial properties against drug-resistant biofilms [58–60]. Previous studies demonstrated that concentrations greater than the MIC are needed to obtain an antibiofilm effect [61, 62]. However, in this study, preformed C. albicans biofilms were significantly dismantled after treatment with the EO and citronellal at the tested concentrations. Interestingly, at 2×MIC and 4×MIC, the EO and citronellal were as effective as chlorhexidine and miconazole in disrupting preformed biofilms, suggesting that they are promising candidates for pharmaceutical formulations, particularly because they have low cytotoxic effects [63].

The motivation to study the anti-Candida potential of C. nardus EO and citronellal was based on their proven activity against other fungal species [22] and a remarkable low toxicity in human epithelial cells. A previous study showed that epithelial cell cultures treated with C. nardus EO at 75 µg/mL showed 75% viability [24, 37], which is a much higher percentage than that proposed in our study. However, further bioavailability and toxicological research is needed to support the clinical application of these bioactive products.

Conclusion

C. nardus EO and its major phytoconstituent, citronellal, showed antifungal activity inhibiting 100% of C. albicans growth at 256 µg/mL. They most likely interact with the cell membrane, but not the cell wall, and did not present any synergistic activity when associated with miconazole or chlorhexidine. Furthermore, C. nardus EO and citronellal effectively inhibited biofilm formation and disrupted preformed biofilms. Collectively, our data provide relevant insights into the potential of these bioactive substances for further clinical testing.

Declarations

Ethics approval

This study complies with the norms of Resolution 466/12 of the National Health Council and was previously approved by the Research Ethics Committee at the Federal University of Pernambuco, under protocol CAAE: 87454417.7.00005208.

Consent to participate

For this type of study, formal consent is not required.

Conflict of interest

The authors declare no competing interests.

Footnotes

Responsible Editor: Melissa Fontes Landell

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Monge RA, Roma’n E, Nombela C, et al. The MAP kinase signal transduction network in Candida albicans. Microbiology. 2006;152:905–912. doi: 10.1099/mic.0.28616-0. [DOI] [PubMed] [Google Scholar]
  • 2.Ramage G, Saville SP, Thomas DP, et al. Candida biofilms: an update. Eukaryot Cell. 2005;4:633–638. doi: 10.1128/EC.4.4.633-638.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Höfs S, Mogavero S, Hube B. Interaction of Candida albicans with host cells: virulence factors, host defense, escape strategies, and the microbiota. J Microbiol. 2016;54(3):149–169. doi: 10.1007/s12275-016-5514-0. [DOI] [PubMed] [Google Scholar]
  • 4.Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence. 2013;4:119–128. doi: 10.4161/viru.22913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Seneviratne CJ, Jin L, Samaranayake LP. Biofilm lifestyle of Candida: a mini-review. Oral Dis. 2008;14(7):582–590. doi: 10.1111/j.1601-0825.2007.01424.x. [DOI] [PubMed] [Google Scholar]
  • 6.Thein ZM, Samaranayake YH, Samaranayake LP. Effect of oral bacteria on growth and survival of Candida albicans biofilms. Arch Oral Biol. 2006;51:672–680. doi: 10.1016/j.archoralbio.2006.02.005. [DOI] [PubMed] [Google Scholar]
  • 7.Kaur S, Soni S, Singh R. Commensal and pathogen: Candida albicans. Ann Geriatr Educ Med Sci. 2017;4:18–21. [Google Scholar]
  • 8.Rajendran R, Sherry L, Nile CJ, et al. Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection—Scotland, 2012–2013. Clin Microbiol Infect. 2016;22(1):87–93. doi: 10.1016/j.cmi.2015.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Ramage G, Martínez JP, López-Ribot JL. Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. 2006;6(7):979–986. doi: 10.1111/j.1567-1364.2006.00117.x. [DOI] [PubMed] [Google Scholar]
  • 10.Tsang CSP, Ng H, McMillan AS. Antifungal susceptibility of Candida albicans biofilms on titanium discs with different surface roughness. Clin Oral Invest. 2007;11(4):361–368. doi: 10.1007/s00784-007-0122-3. [DOI] [PubMed] [Google Scholar]
  • 11.Niimi M, Firth NA, Cannon RD. Antifungal drug resistance of oral fungi. Odontology. 2010;98(1):15–25. doi: 10.1007/s10266-009-0118-3. [DOI] [PubMed] [Google Scholar]
  • 12.Rautemaa R, Ramage G. Oral candidosis—clinical challenges of a biofilm disease. Crit Rev Microbiol. 2011;37(4):328–336. doi: 10.3109/1040841X.2011.585606. [DOI] [PubMed] [Google Scholar]
  • 13.Ungureanu A, Gaman AE, Turculeanu A, et al. Incidence and antifungal susceptibility of Candida albicans infections. Curr Health Sci J. 2016;42(2):164–168. doi: 10.12865/CHSJ.42.02.08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Carmo ES, Lima EO, Souza EL, et al. Effect of Cinnamomum zeylanicum Blume essential oil on the growth and morphogenesis of some potentially pathogenic Aspergillus species. Braz J Microbiol. 2008;39(1):91–97. doi: 10.1590/S1517-838220080001000021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kuriyama T, Williams DW, Bagg J, et al. In vitro susceptibility of oral Candida to seven antifungal agents. Oral Microbiol Immunol. 2005;20(6):349–353. doi: 10.1111/j.1399-302X.2005.00236.x. [DOI] [PubMed] [Google Scholar]
  • 16.Torres SR, Peixoto CB, Caldas DM, et al. A prospective randomized trial to reduce oral Candida spp colonization in patients with hyposalivation. Braz Oral Res. 2007;21(2):182–187. doi: 10.1590/s1806-83242007000200015. [DOI] [PubMed] [Google Scholar]
  • 17.Wingeter MA, Guilhermetti E, Shinobu CS, et al. Microbiological identification and in vitro sensitivity of Candida isolates from the oral cavity of HIV-positive individuals. Rev Soc Bras Med Trop. 2007;40(3):272–276. doi: 10.1590/s0037-86822007000300004. [DOI] [PubMed] [Google Scholar]
  • 18.Menendez A, Li F, Michalek SM, Kirk K. Comparative analysis of the antibacterial effects of combined mouthrinses on Streptococcus mutans. Oral Microbiol Immunol. 2005;20(1):31–34. doi: 10.1111/j.1399-302X.2004.00189.x. [DOI] [PubMed] [Google Scholar]
  • 19.Castro HG, Barbosa ICA, Leal TCAB, et al. Crescimento, teor e composição do óleo essencial de Cymbopogon nardus (L.) Rev Bras Pl Med. 2007;9(4):55–61. [Google Scholar]
  • 20.Beneti SC, Rosset E, Corazza ML, et al. Fractionation of citronella (Cymbopogon winterianus) essential oil and concentrated orange oil phase by batch vacuum distillation. J Food Eng. 2011;102:348–354. [Google Scholar]
  • 21.Man HC, Hamzah MH, Jamaludin H, Abidin ZZ. Preliminary study: kinetics of oil extraction from citronella grass by ohmic heated hydro distillation. APCBEE Proc. 2012;3:124–128. [Google Scholar]
  • 22.Billerbeck VG, Roques CG, Bessière JM, et al. Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger. Can J Microbiol. 2001;47(1):9–17. doi: 10.1139/w00-117. [DOI] [PubMed] [Google Scholar]
  • 23.Li WR, Shi QS, Ouyang YS, et al. Antifungal effects of citronela oil against Aspergillus niger ATCC 16404. Appl Microbiol Biotechnol. 2013;97(16):7483–7492. doi: 10.1007/s00253-012-4460-y. [DOI] [PubMed] [Google Scholar]
  • 24.Zore GB, Thakre AD, Jadhav S, et al. Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of the cell cycle. Phytomedicine. 2011;18(13):1181–1190. doi: 10.1016/j.phymed.2011.03.008. [DOI] [PubMed] [Google Scholar]
  • 25.Delespaul Q, Billerbeck VG, Roques CG, et al. The antifungal activity of essential oils as determined by different screening methods. J Essent Oil Res. 2000;12:256–266. [Google Scholar]
  • 26.Trindade LA, Oliveira JA, Castro RD, et al. Inhibition of adherence of C. albicans to dental implants and cover screws by Cymbopogon nardus essential oil and citronellal. Clin Oral Investig. 2015;19(9):2223–2231. doi: 10.1007/s00784-015-1450-3. [DOI] [PubMed] [Google Scholar]
  • 27.Cleeland R, Squires E (1991) Evaluation of new antimicrobials in vitro and in experimental animal infections In: LORIAN, V. Antibiotics in laboratory medicine 739-787
  • 28.Hadacek F, Greger H. Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochem Anal. 2000;11:137–147. [Google Scholar]
  • 29.Balouri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal. 2016;6(2):71–79. doi: 10.1016/j.jpha.2015.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Frost DJ, Brandt KD, Cugier D, et al. A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J Antibiot. 1995;48(4):306–310. doi: 10.7164/antibiotics.48.306. [DOI] [PubMed] [Google Scholar]
  • 31.Escalante A, Gattuso M, Pérez P, et al. Evidence for the mechanism of action of the antifungal phytolaccoside B isolated from Phytolacca tetramera Hauman. J Nat Prod. 2008;71(10):1720–1725. doi: 10.1021/np070660i. [DOI] [PubMed] [Google Scholar]
  • 32.White RL, Burgess DS, Mandruru M, et al. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard and E-test. Antimicrob Agents Chemother. 1996;40(8):1914–1918. doi: 10.1128/aac.40.8.1914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Mackay ML, Milne K, Goul IM. Comparison of methods for assessing synergic antibiotic interactions. Int J Antimicrob Agents. 2000;15(2):125–129. doi: 10.1016/s0924-8579(00)00149-7. [DOI] [PubMed] [Google Scholar]
  • 34.Balasubramanian D, Schneper L, Merighi M, et al. The regulatory repertoire of Pseudomonas aeruginosa AmpC ß-lactamase regulator AmpR includes virulence genes. PLoS One. 2012;7(3):e34067. doi: 10.1371/journal.pone.0034067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rajasekharan SK, Ramesh S, Satish AS, et al. Antibiofilm and anti-β-lactamase activities of burdock root extract and chlorogenic acid against Klebsiella pneumoniae. J Microbiol Biotechnol. 2017;27(3):542–551. doi: 10.4014/jmb.1609.09043. [DOI] [PubMed] [Google Scholar]
  • 36.Ozcan M, Erkmen O. Antimicrobial activity of the essential oils of Turkish plant spices. Eur Food Res Technol. 2001;212(6):658–660. [Google Scholar]
  • 37.Koba K, Sanda K, Guyon C, Raynaud C, et al. In vitro cytotoxic activity of Cymbopogon citratus L and Cymbopogon nardus L essential oils from Togo. Bangladesh J Pharmacol. 2009;4(1):29–34. [Google Scholar]
  • 38.Kpoviessi S, Bero J, Agbani P, et al. Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin. J Ethnopharmacol. 2014;151(1):652–659. doi: 10.1016/j.jep.2013.11.027. [DOI] [PubMed] [Google Scholar]
  • 39.Wei LS, Wee W. Chemical composition and antimicrobial activity of Cymbopogon nardus citronella essential oil against systemic bacteria of aquatic animals. Iran J Microbiol. 2013;5(2):147–152. [PMC free article] [PubMed] [Google Scholar]
  • 40.Roszaini K, NorAzah MA, Mailina J, et al. Toxicity and antitermite activity of the essential oils from Cinnamomum camphora, Cymbopogon nardus, Melaleuca cajuputi and Dipterocarpus sp. against Coptotermes curvignathus. Wood Sci Technol. 2013;47(6):1273–1284. [Google Scholar]
  • 41.Toledo LG, Ramos MA, Spósito L, et al. Essential oil of Cymbopogon nardus (L.) Rendle: a strategy to combat fungal infections caused by Candida species. Int J Mol Sci. 2016;17(8):1252. doi: 10.3390/ijms17081252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Paraskevas S. Randomized controlled clinical trials on agents used for chemical plaque control. Int J Dent Hyg. 2005;3(4):162–178. doi: 10.1111/j.1601-5037.2005.00145.x. [DOI] [PubMed] [Google Scholar]
  • 43.Gunsolley JC. A meta-analysis of six-month studies of antiplaque and antigingivitis agents. J Am Dent Assoc. 2006;137:1649–1657. doi: 10.14219/jada.archive.2006.0110. [DOI] [PubMed] [Google Scholar]
  • 44.Singh S, Fatima Z, Hameed S. Citronellal-induced disruption of membrane homeostasis in Candida albicans and attenuation of its virulence attributes. Rev Soc Bras Med Trop. 2016;49(4):465–472. doi: 10.1590/0037-8682-0190-2016. [DOI] [PubMed] [Google Scholar]
  • 45.Zore GB, Thakre AD, Jadhav S, et al. Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle. Phytomedicine. 2011;18:1181–1190. doi: 10.1016/j.phymed.2011.03.008. [DOI] [PubMed] [Google Scholar]
  • 46.Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12(4):564–582. doi: 10.1128/cmr.12.4.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Cox SD, Mann CM, Markham JL, et al. The mode of antimicrobial action of essential oils of Melaleuca alternifolia (tea tree oil) J Appl Microbiol. 2000;88(1):170–175. doi: 10.1046/j.1365-2672.2000.00943.x. [DOI] [PubMed] [Google Scholar]
  • 48.Jackson C, Agboke A, Nwoke V. In vitro evaluation of antimicrobial activity of combinations of nystatin and Euphorbia hirta leaf extract against Candida albicans by the checkerboard method. J Med Plant Res. 2009;3(9):666–669. [Google Scholar]
  • 49.Kumar SN, Siji JV, Nambisan B, et al. Activity and synergistic interactions of stilbenes and antibiotic combinations against bacteria in vitro. World J Microbiol Biotechnol. 2012;28(11):3143–3150. doi: 10.1007/s11274-012-1124-0. [DOI] [PubMed] [Google Scholar]
  • 50.Nascimento GGF, Locatelli J, Freitas PC, et al. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol. 2000;31:247–256. [Google Scholar]
  • 51.Ramage G, Rajendran R, Sherry L, et al. Fungal biofilm resistance. Int. J Microbiol. 2012;2012:528521. doi: 10.1155/2012/528521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Di Bonaventura G, Pompilio A, Picciani C, et al. Biofilm formation by the emerging fungal pathogen Trichosporon asahii: development, architecture, and antifungal resistance. Antimicrob Agents Chemother. 2006;50(10):3269–3276. doi: 10.1128/AAC.00556-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Desai JV, Mitchell AP, Andes DR. Fungal biofilms, drug resistance, and recurrent infection. Cold Spring Harb Perspect Med. 2014;1(4):10. doi: 10.1101/cshperspect.a019729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Guiotti AM, Cunha BG, Paulini MB, et al. Antimicrobial activity of conventional and plant-extract disinfectant solutions on microbial biofilms on a maxillofacial polymer surface. J Prosthet Dent. 2016;116(1):136–143. doi: 10.1016/j.prosdent.2015.12.014. [DOI] [PubMed] [Google Scholar]
  • 55.Mathe L, Van Dijck P. Recent insights into Candida albicans biofilm resistance mechanisms. Curr Gen. 2013;59(4):251–264. doi: 10.1007/s00294-013-0400-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Fox EP, Bui CK, Nett JE, et al. An expanded regulatory network temporally controls Candida albicans biofilm formation. Mol Microbiol. 2015;96(6):226–239. doi: 10.1111/mmi.13002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Taff HT, Mitchell KF, Edward JA, et al. Mechanisms of Candida biofilm drug resistance. Future Microbiol. 2013;8(10):1325–1337. doi: 10.2217/fmb.13.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Devkatte AN, Zore GB, Karuppayil SM. Potential of plant oils as inhibitors of Candida albicans growth. FEMS Yeast Res. 2005;5(9):867–873. doi: 10.1016/j.femsyr.2005.02.003. [DOI] [PubMed] [Google Scholar]
  • 59.Tampieri MP, Galuppi R, Macchioni F, et al. The inhibition of Candida albicans by selected essential oils and their major components. Mycopathologia. 2005;159(3):339–345. doi: 10.1007/s11046-003-4790-5. [DOI] [PubMed] [Google Scholar]
  • 60.Agarwal V, Lal P, Pruthi V. Prevention of Candida albicans biofilm by plant oils. Mycopathologia. 2008;165(1):13–19. doi: 10.1007/s11046-007-9077-9. [DOI] [PubMed] [Google Scholar]
  • 61.Oliveira MAC, Borges AC, Brighenti FA, et al. Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity. Braz Oral Res. 2017;31:e-89. doi: 10.1590/1807-3107BOR-2017.vol31.0089. [DOI] [PubMed] [Google Scholar]
  • 62.Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol. 2003;11(1):30–36. doi: 10.1016/s0966-842x(02)00002-1. [DOI] [PubMed] [Google Scholar]
  • 63.Cunha BG, Duque C, Caiaffa KS, et al. Cytotoxicity and antimicrobial effects of citronella oil (Cymbopogon nardus) and commercial mouthwashes on S. aureus and C. albicans biofilms in prosthetic materials. Arch Oral Biol. 2020;109:104577. doi: 10.1016/j.archoralbio.2019.104577. [DOI] [PubMed] [Google Scholar]

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